Destructions of Microorganisms
  Chemical Antimicrobial Agents
    Antimicrobial Agent Susceptibility Testing Resistance
      Agar Disk Diffusion Method
      Broth Dilution Method Determining Minimum Inhibitory Concentration
      Bacterial Resistance to Antimicrobial Agents: Enzymatic
      Bacterial Resistance to Antimicrobial Agents: Mutation

Disinfection is defined as the destruction of pathogenic microorganisms (not necessarily all microbial forms). It is a process involving chemical interactions between a toxic antimicrobial substance and enzymes or other constituents of microbial cells. A disinfectant must kill pathogens while it is in contact with them, so that they cannot grow again when it is removed. In this case it is said to be cidal (lethal), and it is described, according to the type of organism it kills, as bactericidal, virucidal, sporicidal, or simply germicidal. If the antimicrobial substance merely inhibits the organisms while it is in contact with them, they may be able to multiply again when it is removed. In this case, the agent is said to have static activity (it arrests growth) and may be described as bacteriostatic, fungistatic, or virustatic, as the case may be. According to its definition, a chemical disinfectant should produce irreversible changes that are lethal to cells.

Microorganisms of different groups are not uniformly susceptible to chemical disinfection. Tubercle bacilli are more resistant than most other vegetative bacteria because of their waxy cell walls, but of all microbial forms, bacterial endospores display the greatest resistance to both chemical and physical disinfecting agents. Fungal conidia (spores) are also somewhat resistant, although yeasts and hyphae (nonsporing fungal structures), like bacteria, succumb quickly to active disinfectants. Many bactericidal disinfectants also kill viruses, but the viral agents of hepatitis are very resistant. Since microorganisms differ in t

heir response to chemical antimicrobial agents, the choice of disinfectant for a particular purpose is guided in part by the type of microbe present in the contaminated material. Disinfectants that effectively kill vegetative bacteria may not destroy bacterial endospores, fungal conidia, tubercle bacilli, or some viruses. Other practical factors to consider when choosing a disinfectant include the exposure time and concentration required to kill microorganisms, the temperature and pH for its optimal activity, the concentration of microorganisms present, and the toxicity of the agent for skin or its effect on materials to be disinfected.

Purpose To study the activity of some disinfectants and to learn the importance of time, germicidal
concentration, and microbial species in disinfection
Materials Nutrient agar plates
Sterile, empty tubes
Sterile 10-ml pipettes (cotton plugged)
Sterile 1.0-ml pipettes (cotton plugged)
Bulb or other aspiration device for pipette
5% sodium hypochlorite (bleach); 0.05% sodium hypochlorite
Absolute alcohol, 70% alcohol
3% hydrogen peroxide
1% Lysol, 5% Lysol
Iodophor (Betadine)
Antiseptic mouthwash
24-hour nutrient broth culture of Escherichia coli
Three- to six-day-old broth culture of Bacillus subtilis

Table 14.1 summarizes the properties of some common disinfectants. Note that the usedilution (concentration over which the chemical agent is effective) varies among agents and even for the same agent. The disinfectants are categorized as having a low, intermediate, or high level of activity according to the range of microorganisms that they inactivate. Only high-level agents have an effect on resistant bacterial endospores, but all are effective against bacterial vegetative cells and some types of viruses.

Some Common Disinfectants with Their Use-Dilutions and Properties
Table 14.1 Some Common Disinfectants with Their Use-Dilutions and Properties

*Inactivates all indicated microorganisms with a contact time of 30 min or less, except bacterial endospores, which require 6–10 hours contact time. Abbreviations: Int, intermediate; CS, chemical sterilant; , yes; –, no; , variable results. Source: Modified from Rutala W. A. 1996. Selection and Use of Disinfectants in Health Care, pp. 913–936. In C. Glen Mayhall, ed. Hospital Epidemiology and Infection Control. Williams & Wilkins, Baltimore. Modified from Laboratory Biosafety Manual, Geneva: World Health Organization, 1983.

  1. Select one of the chemical agents provided. Pipette 5.0 ml of the solution into a sterile test tube.
  2. To the 5 ml of disinfectant, add 0.5 ml of the E. coli culture. Gently shake the tube to distribute the organisms uniformly. Note the time.
  3. Divide a nutrient agar plate into four sections with a marking pen or pencil. At intervals of 2, 5, 10, and 15 minutes, transfer one loopful of the disinfectant-culture mixture to a section of the nutrient agar plate. Label each plate with the name of the organism, the disinfectant, and its concentration (e.g., E. coli, 1% phenol). Label each section of the plate with the time of exposure (e.g., 2 minutes, 5 minutes, etc.).
  4. Using the same concentration of the same disinfectant, repeat steps 1 to 3 with the culture of B. subtilis.
  5. Inoculate one-half of a nutrient agar plate directly from the E. coli culture and the other half from the B. subtilis culture. Label each half with the name of the organism and the word Control.
  6. Incubate all tubes at 35°C for 48 hours.

  1. Observe all plate sections for growth (+) or absence of growth (−). Complete the table by recording your own and your neighbors’ results with each disinfectant.

  2. *Check label of mouthwash bottle; fill in concentration of active ingredient.

  3. State your interpretation of these results: